During infections by pathogens, such as viruses, bacteria, and parasites that invade human cells, an important component of the immune defense against infection are natural killer (NK) cells. NK cells are a group of lymphocytes that differ from B and T lymphocytes by responding much faster to infections. Unlike B and T cells, NK cells do not express antigen-specific receptors. Therefore, proliferation and activation of NK cells does not begin with a few rare NK clones but with a large number of NK cells. Various chemokines and cytokines, such as type I interferons or interleukin (IL)-12, which are produced early during infections, activate NK cell responses. NK cells exert their function in two ways: by producing cytokines, such as interferon-gamma, and by killing infected cells. NK cells can also kill tumor cells. Despite the importance of NK cells in the innate response to many types of pathogens, it is still unclear what receptors and what signal transduction pathways control their activation. NK cell inhibitory receptors that recognize surface molecules called major histocompatibility complex class I (MHC class I) prevent the killing of normal, healthy cells. The major goal of this project is to define the receptor-ligand interactions that regulate cytotoxicity by NK cells. Cytotoxicity of human NK cells is activated by receptors that bind ligands on target cells, but the relative contribution of the many different activating and inhibitory NK cell receptors is difficult to assess. Using Drosophila insect cells, which express ligands of human NK cell receptors, we show that target cell lysis by resting NK cells is controlled by different receptor signals for cytolytic granule polarization and degranulation. ICAM-1 on insect cells was sufficient to induce polarization of granules, but not degranulation, in resting NK cells. Conversely, engagement of the Fc receptor CD16 by rabbit IgG on insect cells induced degranulation without specific polarization. Lysis by resting NK cells occurred when polarization and degranulation were induced by the combined presence of ICAM-1 and IgG on insect cells. Engagement of receptor 2B4 by CD48 on insect cells induced weak polarization and no degranulation. However, co-engagement of 2B4 and CD16 by their respective ligands resulted in granule polarization and cytotoxicity in the absence of LFA-1?mediated adhesion to target cells. These data show that cytotoxicity by resting NK cells is tightly controlled by separate or co-operative signals from different receptors for granule polarization and degranulation. Freshly isolated, resting natural killer (NK) cells are generally less lytic against target cells than in vitro IL-2?activated NK cells. To investigate the basis for this difference, the contribution of several receptors to activation of human NK cells was examined. Target cell lysis by IL-2?activated NK cells, in a redirected, antibody?dependent cytotoxicity assay was triggered by a number of receptors. In contrast, cytotoxicity by resting NK cells was induced only by CD16, and not by NKp46, NKG2D, 2B4 (CD244), DNAM-1 (CD226), or CD2. Calcium flux in resting NK cells was induced with antibodies to CD16 and, to a weaker extent, antibodies to NKp46 and 2B4. Whereas NKp46 did not enhance CD16?mediated calcium flux, it synergized with all other receptors. 2B4 synergized with three other receptors, NKG2D and DNAM-1 each synergized with two other receptors, and CD2 synergized with NKp46 only. Resting NK cells were induced to secrete TNF-alpha and IFN-gamma, and to kill target cells by engagement of specific, pairwise combinations of receptors. Therefore, natural cytotoxicity by resting NK cells is induced only by mutual co-stimulation of non-activating receptors. These results reveal distinct and specific patterns of synergy among receptors on resting NK cells.